A proteinaceous bone morphogenetic factor was discovered to be present in the bone matrix (Science 150, pp.893-899, 1965) and was named as “bone morphogenetic protein” (hereinafter abbreviated as BMP). Recently, cloning of plural BMP-related genes has been attempted and it has been found that all of them (except BMP-1) belong to the transforming growth factor-β (hereinafter abbreviated as TGF-β) superfamily. Recombinants of some of these factors have been produced by means of a genetic engineering technology and they have been confirmed to have a bone morphogenetic activity, from which their application to the treatment of bone diseases is expected.
Of these factors, the human MP52 (GDF-5, CDMP-1) recently discovered and belonging to the human BMP family (Biochem. Biophys. Res. Commun., 204, pp. 646-652, 1994) has been confirmed by animal tests to be effective as a bone morphogenetic factor. Beyond cartilage and bone morphogenesis MP52 is known to be a multifunctional growth factor effective for example in angiogenesis (WO 95/04819, Yamashita et al. (1997) Experimental Cell Research 235, 218-226), neuronal diseases (WO 97/03188), periodontal and dental applications (WO 95/04819), connective tissue such as tendon and ligament (WO 95/04819, Rickert et al. (2001), Growth Factors 19, 115-126, Wolfman et al. (1997), Journal of Clinical Investigation 100 (2), 321-330) and skin related disorders such as wound healing or hair growth disorders (WO 02/076494). It has been technically reviewed to carry forward the large-scale production thereof by expression using recombinant Escherichia coli (E. coli).
However, when expressed in a large scale in E. coli and others, for instance, when the protein is produced at an amount of several grams per liter of cultured broth, the desired protein generally tends to form an inactive and insoluble inclusion body. This inclusion body comprises monomeric unfolded MP52 and, in order to obtain a dimer or a monomer which is active as a bone morphogenetic factor, the inclusion body must be solubilized, renatured and oxidized to a dimer or monomer of a defined three-dimensional structure (the procedure generally called “refolding”), separated and purified to obtain the desired protein.
The active form of MP52 has the following or the like problems:                1) because of its low solubility in an aqueous solution, it should be handled in the presence of a denaturing agent or under acidic conditions,        2) the protein used for separation tends to nonspecifically adsorb onto a resin for liquid chromatography and bind strongly to media such as ion-exchange or gel filtration, and        3) the surfactant essential for refolding tends to disturb separation, and thus it has been very difficult to establish a process for the purification thereof.        
The purification process recently developed for obtaining a single active form of dimeric MP52 (WO 96/33215, see examples) comprises the following steps:                1. solubilizing an inclusion body by a denaturing agent,        2. separation by ion exchange chromatography in the presence of 6 M urea,        3. sulfonation,        4. separation by gel filtration chromatography in the presence of 6 M urea,        5. refolding in the presence of gluthatione,        6. recovery by isoelectric precipitation, and        7. separation by reverse-phase chromatography.        
The purification process recently developed for obtaining a single active form of refolded monomeric MP52 (WO 99/61611, example 3) resembles that of the dimeric form and comprises the following steps:                1. solubilizing an inclusion body by a denaturing agent,        2. separation by ion exchange chromatography, in the presence of 6 M urea,        3. separation by gel filtration chromatography, in the presence of 6 M urea,        4. refolding in the presence of gluthatione,        5. recovery by isoelectric precipitation,        6. separation by r verse-phase chromatography,        7. isoelectric precipitation.        
The WO 01/11041 also describes monomeric MP52 and its use and contains a purification process in the examples with the following steps:                1. solubilizing an inclusion body by a denaturing agent,        2. separation by reverse-phase chromatography,        4. refolding in the presence of gluthatione,        5. recovery by isoelectric precipitation,        6. separation by reverse-phase chromatography.        
However, the above mentioned processes if scaled up industrially has have encountered some or all of the following and the like problems:                1) a large amount of a denaturing agent is used in order to solubilize the MP52 inclusion body and during purification steps using ion exchange or gel filtration columns because even the unfolded monomer needs constant presence of chaotropes such as 6 M urea, whereby modification of the protein (for example, carbamylation reaction in the case of urea) may be induced,        2) one or more purification steps are performed after solubilization prior to the refolding reaction, in part using an expensive resin for chromatography, especially, for gel filtration chromatography, such as Sephacryl S-20OHR or Superdex 200pg (all available from Pharmacia Biotech) is used in a large amount,        3) a reagent used in refolding, inter alia, oxidized glutathione essential for the refolding reaction is extremely expensive, and        4) when isoelectric precipitation is carried out after the refolding reaction, a dilution is necessarily performed to decrease the concentration of detergent, thus the volume of the solution is increased.        